For many genes, alternative splice site selection is a determinative regulatory event for the expression of divergent protein isoforms. In most cases, alternative splicing pathways are regulated according to cell type and/or developmental stage. In the heart, alternative splicing is a common mechanism by which multiple muscle specific protein isoforms are generated. In many cases, expression of these protein variants is associated with functional, developmental and pathological changes. Therefore, understanding the factors which regulate alternative splice site selection is pertinent and important to understanding the genetic control of heart function and disease. The avian gene encoding the cardiac isoform of the striated muscle thin filament protein, troponin T (cardiac TNT or cTNT) is an ideal model system for investigating the molecular details of pre-mRNA alternative splicing. A single cTNT pre-mRNA precursor is processed along one of two pathways in which a single 30 nucleotide coding exon (exon 5) is either included into or excluded from mature mRNA. Furthermore, selection of cTNT alternative splicing patterns is under tight developmental regulation such that exon inclusion predominates in the early embryonic heart and exon exclusion predominates in the adult. The resulting transition in protein isoforms may alter contractility of heart muscle as the two isoforms lead to different overall responses of the thin filament to calcium concentration. To investigate cTNT alternative splice site selection in vivo, I have established a transient transfection system using cTNT minigenes. This analysis has been used to limit cis-acting elements required for alternative splicing to three small regions of the pre-mRNA and specific sequences within two of these regions have been identified. In this proposal, I will address the molecular mechanism of regulated alternative splice site selection. The established system for transfection analysis of site-specific mutations will be used to complete the systematic analysis of the cTNT pre-mRNA for the cis elements required for alternative splicing. The effects of these mutations will be compared in three cell lines which differentially regulate the selection of the two alternative splicing pathways. This approach will not only define the elements required for alternative splicing, it will also characterize the response of these elements to trans acting factors that regulate splice site selection. The identification of all cis elements required for alternative splicing of the cTNT pre-mRNA should provide tentative information about the regulatory mechanism. In the second phase of the project, I will analyze in detail the role of these elements in regulated splice site selection. A cell-free system will be used to address several questions including: i) Do mutations that block alternative splicing in vivo interfere with the binding of the splicing machinery components to the alternative splice sites? ii) Does the sequence affected by the mutation directly and specifically bind to nuclear factors? and iii) Does binding activity differ in nuclear extracts from cells that differentially regulate splice site section? The overall and long-term goal of these experiments is to determine the molecular mechanisms by which pre-mRNA alternative splicing is regulated during heart development.